Patricia R. Slev

Pathogen‐Based Models Favoring MHC Genetic Diversity

We present six models that are currently the most likely ways that pathogens might favor the evolution of MHC genetic diversity. Although each model makes one or more unique predictions, the current lack of crucial data prevents distinguishing the relative importance of each model. However, this first-time organization of these models should contribute to the design of critical experiments. This synthetic review yields at least three essentially new ideas. First, MHC-dependent immune recognition may be sufficiently redundant to render it essentially escape-proof by pathogens. Second, the four models based on pathogen escape do not work (or work weakly) for diversifying class II genes, unless class II-restricted cytotoxic T-cells are important, an idea that is controversial. Third, pathogen-escape events have traditionally been thought to result in only frequency-dependent selection but here we show that heterozygote advantage is an inevitable consequence of such pathogen evasion. Therefore, the controversy over the relative importance of these two forms of balancing selection is largely a false dichotomy.

Experimental viral evolution to specific host MHC genotypes reveals fitness and virulence trade-offs in alternative MHC types

The unprecedented genetic diversity found at vertebrate MHC (major histocompatibility complex) loci influences susceptibility to most infectious and autoimmune diseases. The evolutionary explanation for how these polymorphisms are maintained has been controversial. One leading explanation, antagonistic coevolution (also known as the Red Queen), postulates a never-ending molecular arms race where pathogens evolve to evade immune recognition by common MHC alleles, which in turn provides a selective advantage to hosts carrying rare MHC alleles. This cyclical process leads to negative frequency-dependent selection and promotes MHC diversity if two conditions are met: (i) pathogen adaptation must produce trade-offs that result in pathogen fitness being higher in familiar (i.e., host MHC genotype adapted to) vs. unfamiliar host MHC genotypes; and (ii) this adaptation must produce correlated patterns of virulence (i.e., disease severity). Here we test these fundamental assumptions using an experimental evolutionary approach (serial passage). We demonstrate rapid adaptation and virulence evolution of a mouse-specific retrovirus to its mammalian host across multiple MHC genotypes. Critically, this adaptive response results in trade-offs (i.e., antagonistic pleiotropy) between host MHC genotypes; both viral fitness and virulence is substantially higher in familiar versus unfamiliar MHC genotypes. These data are unique in experimentally confirming the requisite conditions of the antagonistic coevolution model of MHC evolution and providing quantification of fitness effects for pathogen and host. These data help explain the unprecedented diversity of MHC genes, including how disease-causing alleles are maintained.

Intermethod differences in results for total PSA, free PSA, and percentage of free PSA.

Serum prostate-specific antigen (PSA) assays differ in calibration and response to different PSA forms. We examined intermethod differences in total PSA (tPSA) and free PSA (fPSA) measurements. We tested 157 samples with tPSA concentrations of 2 to 10 ng/mL (2-10 microg/L) using 6 PSA/fPSA method pairs and 1 tPSA method: ADVIA Centaur (complexed and total; Siemens Diagnostics, Tarrytown, NY), ARCHITECT i 2000(SR) (Abbott Diagnostics, Abbott Park, IL), AxSYM (Abbott Diagnostics), IMMULITE 2000 (Siemens Diagnostics), Modular E170 (Roche Diagnostics, Indianapolis, IN), UniCel DxI 800 (Beckman Coulter, Brea, CA), and VITROS ECi (tPSA only; Ortho-Clinical Diagnostics, Raritan, NJ). Regression analysis was performed for PSA, fPSA, and percentage of fPSA with the ARCHITECT i 2000(SR) comparison method. Differences between test and comparison methods were estimated at 2.5, 4.0, and 10.0 ng/mL (2.5, 4.0, and 10.0 microg/L) for tPSA and 15%, 20%, and 25% for percentage of fPSA. Relative differences were more than 10% at 4.0 ng/mL (4.0 microg/L) tPSA for the Centaur, IMMULITE, ECi, and DxI methods. At 20% fPSA, the relative difference was more than 10% for all methods except the AxSYM. Additional harmonization is needed for tPSA and fPSA methods.

Simultaneous Genotyping of rs12979860 and rs8099917 Variants Near the IL28B Locus Associated with HCV Clearance and Treatment Response

Recent genome-wide association studies have identified two host single-nucleotide polymorphisms (SNPs) near the IL28B gene (rs12979860 C/T and rs8099917 T/G) that are associated with sustained virological response in patients infected with the hepatitis C virus. Herein, we describe a rapid multiplexed dual-color fluorescence resonance energy transfer (FRET) probe assay that accurately genotypes for both SNPs simultaneously. A single-nucleotide extension assay was also developed for verification of genotypes. Agreement (100%) was observed in genotype calls between the FRET and single-nucleotide extension methods for both SNPs, yielding 100% analytical sensitivity and specificity. By using the FRET assay, 443 samples of varying ethnic backgrounds were genotyped and six different compound genotypes (rs12979860/rs8099917) were detected in whites, Asians, Middle Easterners, Hispanics, and African Americans, at the following frequencies: CC/TT (39.2%, 78.9%, 40.0%, 33.9%, and 16.8%), CT/TT (20.8%, 0%, 40%, 9.3%, and 37.0%), TT/TT (2.4%, 0%, 0%, 3.4%, and 35.3%), CT/TG (24.0%, 19.7%, 20%, 39.8%, and 3.4%), TT/TG (8.0%, 1.4%, 0%, 3.4%, and 5.9%), and TT/GG (5.6%, 0%, 0%, 10.2%, and 1.7%), respectively. The multiplexed FRET assay can be used to effectively genotype for both SNPs in a single tube, with high analytical sensitivity and specificity.

Copy Number Variation and Incomplete Linkage Disequilibrium Interfere with the HCP5 Genotyping Assay for Abacavir Hypersensitivity

Carriers of HLA-B*57:01 are at risk for Abacavir hypersensitivity reaction (ABC-HSR). In Caucasians, a SNP (rs2395029) in the HCP5 gene is reported to be in linkage disequilibrium (LD) with HLA-B*57:01. Genotyping the HCP5 SNP has increasingly been adopted as a simple method to screen for susceptibility to ABC-HSR. We genotyped both the HCP5 SNP and HLA-B*57:01 in a set of 1888 samples and found a good correlation; significantly, however, one HLA-B*57:01-positive sample tested negative for the HCP5 SNP. In addition, HCP5 could not be amplified in two samples, both negative for HLA-B*57:01. Further investigation demonstrated both samples were homozygous for deletion of the HCP5 gene. The fact HCP5 occurs within a region of copy number variation and the fact LD is incomplete and may vary between ethnicities should be considered when using the HCP5 SNP as a surrogate marker for HLA-B*57:01.

Efficacy of the Detection of the α1-Antitrypsin “Z” Deficiency Variant by Routine Serum Protein Electrophoresis

Deficiency of alpha1-antitrypsin (AAT) is a common but underdiagnosed genetic disorder. Severe AAT deficiency may be detected by the absence of alpha1-globulin protein fraction by serum protein electrophoresis (SPEP). Routine SPEP may represent an underused resource for the identification of AAT deficiency. Total alpha1-globulin protein was measured in 47 MM, 24 MZ, and 19 ZZ phenotype serum samples by a Sebia CAPILLARYS (Norcross, GA) capillary electrophoresis system. Measured serum AAT concentrations by immunoassay exhibited moderate correlation with measured SPEP alpha1-globulin fraction concentrations. In this sample set, 16 (84%) of the ZZ, 7 (29%) of the MZ, and none of the MM sample phenotypes exhibited alpha1-globulin concentrations of less than 0.21 g/dL. From estimates of MZ and ZZ phenotype prevalence, it can be calculated that 1 ZZ phenotype should be present in approximately every 31 samples with alpha1-globulin concentrations of less than 0.21 g/dL. Clinicians should consider investigation of potential AAT deficiency in patients who exhibit low alpha1-globulin protein levels by routine SPEP.

Profound T-cell lymphopenia associated with prenatal exposure to purine antagonists detected by TREC newborn screening

Recognition of non-SCID and secondary causes of T cell lymphopenia detected by TREC newborn screening is important in directing subsequent care and identifying those who would not benefit from more invasive interventions. Here, we report two infants with low TRECs and severe, but self-resolving, T cell lymphopenia identified by SCID NBS that were caused by in utero exposure to purine antimetabolites.

Thymic output: Assessment of CD4+ recent thymic emigrants and T‐Cell receptor excision circles in infants

CD4+ recent thymic emigrants (CD4+ RTEs) constitute a subset of T cells recently generated in the thymus and exported into peripheral blood. CD4+ RTEs have increased copy numbers of T‐cell receptor excision circles (TREC). They are characterized by the expression of CD31 on naïve CD4 T‐cells. We aimed to validate a flow‐cytometry assay to enumerate CD4+ RTEs and assess its performance in relation to TREC measurement.

Comparison of Enzyme Immunoassays for Detection of Antibodies to Hepatitis D Virus in Serum

ABSTRACT Serology remains critical for diagnosing hepatitis D virus (HDV) infection, which affects 15 to 20 million people worldwide, but the literature on characterizing commercial enzyme immunoassays (EIAs) dates back to 15 years ago. We evaluated 2 commercial EIAs currently available for detecting anti-HDV antibodies. The DiaSorin assay demonstrated 100% sensitivity and specificity. Using a modified cutoff value, the Cusabio assay demonstrated a sensitivity of 81.3% and specificity of 90.9%. Our data show that recently developed EIAs are reliable for anti-HDV antibody detection.

Host Factor Pharmacogenetics for Treatment of Infectious Diseases

Progress in therapeutics and testing for infectious diseases over the past 100 years has resulted in dramatic reductions in morbidity and mortality. However, with developments of effective drugs has come the recognition that not all individuals respond the same to a given therapy, and this marked the beginning of initiatives to study human genetic variations as a factor in response to drug therapy. The pharmacogenetics of infectious diseases is in its infancy; however, the goal of maximizing drug efficacy while minimizing toxicity has important implications for clinical care and better understanding of the biology of the host-pathogen interface. This chapter provides an overview of the progress that has been made in pharmacogenetics and summarizes the challenges that remain for bridging the gap between research and clinical use. The few illustrative examples of genetic testing currently utilized as tools for patient management are discussed.